This invention relates to methods of separating the alpha, beta, gamma and delta (.alpha., .beta., .gamma. and .delta. respectively) isomers of tocopherol from mixtures containing these isomers. This invention is especially significant in that it is now possible to achieve a complete separation of the gamma and beta forms which heretofore had not been truly separated. Moreover, this invention provides a quick and simple means for making such a separation without the need for complicated derivatization reactions or other destructive techniques. This invention is very significant with respect to vitamin E chemistry in that all the naturally occurring forms of this vitamin (i.e. the tocopherol isomers) can be separated and isolated.
Within the last decade, the group of naturally occurring compounds possessing vitamin E activity has been shown to include alpha-tocopherol (5,7,8-trimethyltocol), beta-tocopherol (5,8-dimethyltocol), gamma-tocopherol (7,8-dimethyltocol) and delta-tocopherol (8-methyltocol). The structures of these compounds are given below for comparative and illustrative purposes as formulas I, II, III and IV respectively. ##STR1##
The term "vitamin E" originally denoted a partially characterized material in vegetable oils that was found to be essential for the rat to maintain fertility. First discovered in 1922, it was found that more than one naturally occurring substance and several synthetic compounds acted like, or had some effect upon vitamin E deficiency symptoms in the body. These effects, more specifically, the symptoms of vitamin E deprivation were discovered to vary according to the particular species of animal involved. As a result of the lack of knowledge as to the actual composition, vitamin E "activity" was used for many years to designate the amount of dosage required for a particular agent to cure a particular deficiency symptom.
Finally, four naturally occurring compounds having vitamin E activity were isolated and identified. These substances were designated as .alpha., .beta.-, .gamma.-, and .delta.-tocopherol. Chemically, all four are methyl derivatives of tocol[2-methyl-2-(4',8',12'-trimethyltridecyl)-6-chromanol]. Simply, the structures are methyl derivatives of a chromane ring type structure of the general formula ##STR2## wherein R is 4,8,12-trimethyl-n-tridecane, and R.sub.1 is methyl or hydrogen where at least one R.sub.1 is methyl. Additional compounds analogous to the tocopherols have also been characterized. These compounds are the methyl derivatives of tocotrieno[2-methyl-2-(4',8',12'-trimethyltrideca-3',7',11'-trienyl)-6-chr omanol]. The main difference structurally in the latter compounds is that they contain three unsaturated bonds in the side chain but are otherwise of the same general formula as (V) except that R would be the formula ##STR3##
Although the tocopherols and tocotrienols appear to be of rather similar chemical structure, they have been found to exhibit markedly different biological properties. In fact, distinct differences in bioactivity have been noted for the different isomers of tocopherol alone. Alpha-tocopherol, with its completely methylated ring and saturated side chain, possesses the highest biological activity. Because of this high potency, the term ".alpha.-tocopherol" is now gaining wide use as an identification of "vitamin E."
Alpha-tocopherol and its acetate are the forms most used commercially. The naturally occurring d form is the most active isomer physiologically with the racemic synthetic dl-.alpha.-tocopherol and its esters being less potent on a weight for weight basis than the d form. Alpha-tocopherol acetate is the principal commercial form of vitamin E in medicine. Additional uses for the tocopherols include, in food technology, their use as antioxidants to retard rancidity in fatty materials. These compounds have also, with some degree of notariety, been employed as actives for aerosol deodorants. Additional uses include other cosmetics including "coldcreams." Because of their antioxidant activity, these compounds are theorized to have possible anticancer activity especially in those forms suspected of being caused by free radical initiation. This particular activity is somewhat speculative due to lack of knowledge or at least hard data in this field.
The tocopherols are found distributed in many foods in an unesterified form. The highest concentrations are found in the cereal grain oils. Crude corn and wheat oils for example may contain 200 mg of tocopherol per 100 g of oil. There seems to be, however, great variation with respect to what particular oil is used. Certain vegetable oils, such as coconut oil, are practically devoid of tocopherols. Similarly, the proportion of the various isomers also varies widely. For example, about 90% of the tocopherol in safflower oil is .alpha.-tocopherol whereas only about 20% of corn and soybean oil is in the alpha form. The gamma form predominates in corn oil, whereas both the gamma and delta forms predominate in soybean oil. Wheat oils on the other hand have mixtures of tocopherol with up to 65% in the beta form.
Significant losses of vitamin E may occur during the processing and cooking of foods. The degree of loss is dependent of course upon the mode of process, etc. The amount of tocopherol left in refined salad oil, for example, depends upon the severity of the refining process.
A detailed discussion of vitamin E, its properties, occurrence, isolation and synthesis, assay, functions and uses can be found in the Encylcopedia of Chemical Technology, Kirk and Othmer, Volume 21, pages 574 to 585, which is incorporated herein by reference.
A serious disadvantage of all currently used methods for the separation and determination of tocopherols in food substances, in particular vegetable oils and products made therefrom, is that there occur significant losses and destruction of the isomers during the steps of the preparation and during the final quantitation. Additionally, and even more significantly, the beta-and gamma-tocopherol isomers have not satisfactorily been separated from mixtures containing these very similar dimethyl forms.
Slover et al., "Journal American Oil Chemists' Society", 44(3), 161-166, 1967, describe a gas-liquid chromatographic method for the identification and estimation of the individual tocopherols as their trimethylsilyl (TMS) ethers, after purification of the unsaponifiable matter by thin-layer chromatography (TLC). Comparison of the chromatograms of these TMS ethers for soybean oil and for wheat germ oil tocopherols indicates that this technique does not separate the gamma and beta isomers, as the gamma-tocopherol found in soybean oil has the same retention time as does the beta-tocopherol found in wheat germ oil. This procedure was investigated by the applicant and results obtained therein support the observation of lack of separation of these isomeric forms.
Lovelady, "Journal of Chromatography", 85, 81-92, 1973, as well as Lehmann, "Lipids", 6(1), 35-39, 1971, determined the individual tocopherols in plasma and red blood cells. Lovelady's method is similar to Slover's in that it involves extraction, purification of unsaponifiables by TLC, derivatization of the tocopherols, and quantitation by GLC. Lovelady reports retention times (relative to a 5,7-dimethyltocol internal standard as 1.00) for beta-tocopherol of 0.81 and for gamma-tocopherol as 0.82. Realistically, these retention times are not indicative of true separation. Moreover, for quantitation, skillful physical removal of the beta- or gamma- isomer zone, as well as the respective interface zone where the isomers intermingle from the TLC plate is required.
"Method of Analysis-AOAC", 11th Edition, Volume 54, 1971, and Nelson et al. "Journal American Oil Chemists' Society", 47(8), 259-261, 1970, discuss the analysis of tocopherols in vegetable oil, and soya sludges and residues respectively. The former is not only excessively time consuming but also results in considerable tocopherol losses. Nelson, with the advantage of dealing with the high levels of tocopherols found in sludges, was unable to separate the beta and gamma forms.
Feeter, "Journal American Oil Chemists' Society", 51(4), 184-187, 1974, determined total tocopherols in vegetable oil distillates by the Emmery-Engle reaction and the individual tocopherols as their propionate esters by GLC.
Slover, "Lipids", 6(5), 291-296, 1971, and "Journal American Oil Chemists' Society", 46(8), 417-420, 1969, reported data of tocopherol content in foods and fats, using TLC, derivatization and GLC. Christie et al., "Analyst", 98, 161-167, 1973, reported vitamin E content in food, using colorimetry for total tocopherols, and GLC for the individual isomers; again without separating the beta- and gamma-tocopherols.
Niederstebruch and Hinsch, "Fette Seifen Anstr. Mittel", 69(8), 559-563, 1967, describe determinations of tocopherols via a polarographic technique, which requires oxidation of the tocopherols to the tocopherylquinones.
Wachs and Melchert, "Deutche Lebensmittel-Rundschau", 67(7), 221-225, 1971, disclose a method wherein the unsaponifiable tocopherol is acetylated prior to quantitative analysis by GLC. Following acetylation, the esters are first refined by open-column chromatography on Sephadex LH-20 and then analyzed by GLC.